Introduction: For decades, scientists have attempted to develop models for growing and studying primary tumors outside of the human body. Even today, one of the greatest challenges in cancer research has been generation of stable cancer cell lines from primary tumors. Most primary cell cultures, regardless of the method used to generate them, suffer from limited lifespan due to cellular senescence. Cultured cells transformed with SV40 virus large T antigen or over-expression of hTERT has been limited by genomic variation and other changes that vary with the passage number. Recently, a new approach was described by the Schlegel group at Georgetown (Liu et al, AJP 2012) called “conditionally reprogrammed cells” (CRCs). They showed these cells (derived from primary tumors or normal tissue) could be grown indefinitely under defined conditions without using any extrinsic genetic immortalization technique. These cells have been shown to maintain the karyotype similar to the tissue of origin even after prolonged passaging. Here we show that we can obtain confirmatory results at a separate institution and further show that in lung cancer, the CRC lines maintain histotype specific characteristics.

Method: The CRC technique requires mouse irradiated J2 cells as feeder cells and a ROCK inhibitor to rapidly grow and expand patient's epithelial cells. We have grown cultures from six tissue types including lung, breast, pancreas, kidney, colon, and salivary gland. All tissue samples were collected by Yale University Pathology Tissue Services and processed in our lab for generating CRC lines. We have characterized lung CRC lines for morphology and immunophenotyping using H&E stains and histological markers including CK7, p63 and TTF1. We have also performed gene expression profiling using Illumina bead arrays on selected lung and pancreatic samples.

Results: We have successfully established CRC lines from all six tissue types. Our success rate exceeds 70% for lung and breast tumor samples while other tissues show somewhat lower rates. Even traditionally challenging cells to culture, like pancreatic and salivary gland tumors have been successfully cultured. Preliminary morphological and histological analysis on lung CRC lines show that features of lung adenocarcinoma (ADCA) and lung squamous cell carcinoma (SQCC) are maintained in CRC lines. Gene expression profiling shows that lung CRC lines from SQCC samples clustered together and are separate from ADCA. Profiles from a pancreatic CRC line recapitulated the profile of the corresponding frozen tissue.

Conclusion: This novel CRC technology represents a new approach to culturing cells from tumors and normal cells, including tumors that have been historically challenging to grow. The resulting cells appear highly similar to the tumors from which they were derived. Future studies are needed to further characterize the CRC system and to test its value for assessment of personalized drug sensitivity.

Citation Format: Seema Agarwal, Joanna Hu, Kelly Stanton, Kurt Schalper, Yuval Kluger, Elizabeth Zarrella, Xuefeng Liu, Richard Schlegel, David L. Rimm. Next generation cell line models: conditionally reprogrammed cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1569. doi:10.1158/1538-7445.AM2013-1569